2-Methoxyestradiol (2ME2) is an anticancer agent with antiproliferative, antiangiogenic, and proapoptotic effects. A major proposed mechanism of drug action is the disruption of the microtubule skeleton, leading to the induction of cell cycle arrest and apoptosis. In addition, other mechanisms of action have been proposed, including the generation of reactive oxygen species (ROS), inhibition of hypoxia-inducible factor (HIF), and interference with mitochondrial function. In this study, we used a selection of 2ME2 analogues to conduct structure activity analysis and correlated the antiproliferative and proapoptotic activity of the various analogues with their effects on different drug targets. A good correlation was observed between drug activity and effects on microtubule function. In contrast, our results indicate that effects on ROS, HIF, and mitochondria are unlikely to contribute significantly to the cellular activity of 2ME2. Thus, our data indicate that the structural requirements for inducing ROS and inhibition of complex I of the mitochondrial electron transport chain were different from those required for proapoptotic drug activity. Furthermore, antioxidant treatment or overexpression of catalase did not inhibit the cellular activity of 2ME2 in epithelial cancer cells. Inhibition of HIF required much higher concentrations of 2ME2 analogues compared with concentrations that inhibited cell proliferation and induced apoptosis. Our results thus provide a better insight into the mechanism of action of 2ME2 and reveal structural requirements that confer high cellular activity, which may aid future drug development.